The numbers which appear in brackets throughout the following description refer to the List of References found at the end of the description. Reference [1] is an excellent current review of interferons with articles relating to various aspects of the interferon system. Reference [2] is an older book which reviews interferon research.
Interferons are natural cell products produced by appropriately stimulated vertebrate cells that provide antiviral resistance against many different kinds of viruses. The currently accepted criteria for an interferon include: (a) differentiation from nonspecific toxic effects on cells which would otherwise limit virus growth; (b) inhibitory effects on a range of unrelated viruses; (c) demonstration of intracellular effects requiring protein and ribonucleic acid synthesis; (d) loss of biological activity after proteolytic enzyme treatment e.g., trypsin; (e) relative species specificity; (f) neutralization of its biological activity by specific antiserum; (g) a protein of molecular weight between 12,000 and 45,000, although some have even greater molecular weights; (h) relative stability at low pH; and (i) relatively high specific activity in relation to protein concentration. Specific characteristics of interferons from different animal species or cells may differ from one another.
While it is known that, as against viral infections, interferon acts intracellularly to limit viral synthesis, the mechanism by which this is accomplished has not yet been established precisely. The action of interferon is not to inactivate directly the virus particles, but to make the cell resistant to virus infection in some indirect manner. Interferon appears to bind to cell surface receptors and require cellular metabolic activity for expression of such resistance, especially ribonucleic acid and protein synthesis. Thus, it is generally accepted that the action of interferon in a cell is to induce the synthesis of another protein which appears to act at the ribosomal level to interfere with the synthesis of viral-coded, functional enzymes and structural coat proteins necessary for viral replication. It is generally true that interferon is most active in cells of the same animal species from which it was produced, but instances are known of family or order crossreactivity and are reported in the literature.
Interferon production by a cell, in either a suitable culture or in an intact animal, is stimulated by a variety of agents referred to as "inducers". A large number of animal viruses are known to be effective as interferon inducers, although not all viruses induce interferon production. Examples of viruses effective as interferon inducers are influenza, Newcastle disease, and Sendai viruses. Nonviral interferon inducers are also known, including both natural and synthetic products. The natural products, mostly of microbial origin, include nucleic acids, especially double-stranded RNAs; intracellular microbes such as bacteria, ricksettia and protozoa; and microbial products such as lipopolysaccharides and protein. Some of the synthetic inducers include synthetic RNAs, such as poly rI-poly rC; polymers such as polyacrylic acid and polysulfates; and low molecular weight substances such as cycloheximide, tilorone, and basic dyes. A more complete listing of natural and synthetic nonviral inducers is set forth in Reference [3].
Interferons are said to have been discovered by Isaacs and Lindenmann in 1957 [4; see 5 & 6]. Since that time, there has been considerable research directed towards the investigation of the clinical use of interferons, particularly human interferon. However, this has been inhibited by the limited availability of interferon in sufficient quantities to enable large scale clinical investigation, and it is only within the last few years that the pace of such work has accelerated. Interferon has been demonstrated to be effective in limiting several experimental viral infections of animals, a number of infectious agents have been shown to be inhibited by a variety of materials known to be interferon inducers, and experiments in vitro and with laboratory animals have revealed interferon exhibits antitumor activity; see the literature reviewed in Reference [3]. Reference [7] presents the results of a workshop convened by the National Institute of Allergy and Infectious Diseases and the National Cancer Institute on Mar. 21-23, 1978 which reviewed the information from clinical trials of exogenous interferon in the treatment of both infectious and metastatic diseases. Other of the references attest to the current high degree of interest in the examination of the clinical use of interferon, [8,9]. Effective interferon treatment has been reported for herpesvirus infections, hepatitus B infections and respiratory virus infections, as well as osteogenic sarcoma, lymphoma, breast cancer, multiple myeloma and leukemia. The interest in interferons and the fact that relatively large amounts are required for many clinical uses has created a need for large-scale interferon production methods, which in turn results in a need for an effective method of stabilizing interferons against loss of biological activity during and after production.
A number of techniques for the production of interferons from various types of cells are described in the literature; see e.g. [11, 12, 13 & 14]. In general, the procedures involve the following stages:
(1) Cell collection and purification. Human interferon is produced from leukocytes obtained from blood centers, diploid fibroblasts usually obtained from infant foreskins, and lymphoblastoid cells, generally of the Namalva cell line.
(2) Induction of the cells with a suitable inducer, often preceded by priming with interferon or followed by superinduction in the production of fibroblast interferon.
(3) Incubation of the induced cells in a suitable medium for a sufficient time to produce interferon.
(4) Purification and concentration of the crude interferon. Classical purification procedures such as acid precipitation, dialysis, molecular sieve chromatography, isoelectric focusing, and affinity chromatography have been employed to purify interferons. Purifications, presumably to homogeneity, have been recently reported, for human interferons to 2.times.10.sup.8 units/mg of protein, and for mouse interferon 1.times.10.sup.9 units/mg of protein.
(5) Safety testing of the interferon.
(6) Storage of the interferon prior to and during clinical administration.
The cell cultures are typically incubated in suspension or as a monolayer in roller bottles [12], although recent work has described the use of microcarrier beads electrically charged to attract and hold the cells [8]. At various stages the interferon is subjected to mechanical stress, such as imposed by stirring and filtration, or thermal conditions which can result in inactivation of the interferon; this can often occur during the purification and concentration, safety testing and storage stages. Human fibroblast interferon is particularly subject to inactivation by heat and mechanical stress. Human leukocyte interferon is more stable than fibroblast interferon and has generally been regarded as being a relatively stable molecule, but it is now known that losses in activity especially occur in very dilute solutions containing low concentrations of protein. Our paper [10] and its references contain more detailed information regarding the inactivation of interferons.
The prior art techniques for the stabilization of interferons include the following:
(a) Human leukocyte interferon can be stabilized against thermal inactivation by sodium dodecyl sulfate (SDS). However, SDS binds tenaciously to proteins and is almost impossible to remove, which lessens its clinical usefulness.
(b) Freeze drying of interferons can limit loss of activity during storage. This is useful only for storage and cannot be used during the processing of interferons.
(c) U.S. Pat. No. 3,981,991, Stewart & deSomer, relates to the stabilization of interferons using a combination of three compounds: an agent such as urea for disrupting non-covalent bonds; an agent such as mercaptoethanol for reducing disulfide bonds; and an anionic or cationic surface-active agent such as SDS.
(d) U.S. Pat. No. 4,100,150, Cartwright, relates to the stabilization of interferons with thioctic acid, which acts to reduce sulfhydryl (--SH) groups of the interferon without reducing its disulfide (--S--S--) linkages.
(e) It has been reported that human fibroblast interferon can be stabilized for long term storage at 4.degree. C., with ethylene glycol [22].
We have now found a new technique for interferon stabilization which employs compounds not heretofore known to be effective for stabilizing interferons.